HIC and Bradford Assay Lab Report PDF

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Lab #9 and 10 Report (HIC and Bradford Assay)I. Figures and TablesTable 1. Fluorescent Protein Descriptions Name # of Amino AcidsMW (kPa) pI GRAVY Predicted ElutioneGFP 238 26,884 5 -0 3 rd mOrange 236 26,693 6 -0 2 nd mCherry 236 26,722 5 -0 1 stFigure 1. Hydrophobic Interaction ChromatographyFigur...

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Lab #9 and 10 Report (HIC and Bradford Assay) I.

Figures and Tables Table 1. Fluorescent Protein Descriptions Name # of Amino MW (kPa) Acids eGFP 238 26,884.3 mOrange 236 26,693.2 mCherry 236 26,722.1

pI

GRAVY

5.8 6.03 5.62

-0.524 -0.678 -0.744

Predicted Elution 3rd 2nd 1st

Figure 1. Hydrophobic Interaction Chromatography Nothing

Load

1

eGFP

2

3

mOrange mCherry

4

5

6

7

Figure 1. Hydrophobic Interaction Chromatography Depicted above are the different trials run though the chromatography lab. The four tubes on the top row are the three pure fluorescent proteins. The bottom row shows the serial separation of the different proteins from one another. The first protein to be eluted (shown in number 1 and 2 on the bottom row) was mCherry. The next three tubes shows the elution of mOrange. Finally, the last two tubes show the final elution of eGFP. All of these samples shown are above a blue light, making them fluoresce and easier to see.

Figure 2. Absorbance of BSA Protein

Absorbance of BSA Protein 0.4 0.35

Abs [595nm]

0.3 0.25 0.2 0.15 0.1 0.05 0 0

10

20

30

40

50

60

70

80

µg of BSA Protein

Figure 2. Absorbance vs. Concentration of BSA Protein The figure above shows the plot of the absorbance [595nm] vs. the concentration of BSA protein. Using the Bradford Dye-Method, as the concentration of BSA protein increases, the deeper shade of blue the dye becomes resulting in the increase of the absorbance values. Table 2. Absorbance Values of Fractions From HIC HIC Fraction Volume of Sample Volume of dH2O (µL) (µL) Load 15 85 1 15 85 2 15 85 3 25 75 4 15 85 5 25 75 6 15 85 7 15 85 II.

Absorbance [595nm] 0.16 0.06 0.085 0.061 0.05 0.17 0.07 0.20

Introduction The purpose of the first part of this lab report (Lab 9) is to prepare the materials needed and to carry out a Hydrophobic Interaction Chromatography (HIC). Chromatography is used to separate one compound from another and, in this case, using hydrophobic interactions to separate three fluorescent proteins from a mixture of all three. In Table 1, it shows the number of amino acids present in each protein, the molecular weight of each protein, the optimum pI, the grand average hydropathy, and the predicted elution. Since the GRAVY values of the three proteins differ the most, a hydrophobic interaction chromatography would yield the best results to separate them from one another. The elution series was

determined by the GRAVY values of the proteins; the more negative the GRAVY value, the more likely for it to be eluted out of the solution. The purpose of the second part of this lab report (Lab 10) is to determine the precise amount of protein (concentration) in a solution using the Bradford Dye-binding method. This method is carried out by using a dye (Coomassie Brilliant Blue G-250) which is red-orange when free in solution and blue when bound to protein. The dye will turn different shades of blue depending on the amount of protein present. The more protein there is, the deeper shade of blue the dye will be. The concentration of the protein was then assessed by putting solutions of differing concentrations through a spectrophotometer and taking the absorbance readings of them. This way, it is possible to come up with the concentration of each solution. III.

Materials and Methods For the protein chromatography lab, a variety of differing buffer solutions were used in order to separate the three fluorescent proteins from one another. The first buffer used to separate mCherry from the solution of all three fluorescent proteins was the equilibrium/wash buffer. This buffer comprised of 1.5M of (NH4)2SO4 diluted with dH2O. This buffer was used twice to completely elute the mCherry. The second buffer was the salt elution buffer used to separate mOrange from the remaining two fluorescent proteins. This buffer was comprised of 1.0 M (NH4)2SO4 diluted with dH2O. This buffer solution was used twice to completely elute the mOrange from the eGFP. The final buffer used was a no salt elution buffer comprised of only dH2O to separate eGFP. For the protein quantitation lab, the Bradford Dye-binding method was used to quantify the amount of protein (concentration) in certain solutions. Coomassie Brilliant Blue G-250 dye was used to detect protein in solution. After the reaction went to completion, the solutions were put through a spectrophotometer to read the absorbance and to find the concentration from them.

IV.

Results The purpose of the HIC experiment was to make buffers to carry out a chromatography and then to use these different buffer solutions in order to elute these proteins differing in hydrophobicity one at a time. Figure 1 shows the differing trials of the chromatography and which proteins elute first. The samples are placed over a blue light in order to cause the proteins to fluoresce and to make them more easily visible. Due to the differing hydrophobicity among the three proteins, the order of elution happened to be mCherry first, mOrange second, and eGFP last, thus supporting the original hypothesis. The purpose of the protein quantification experiment was to use the Bradford Dye-Method to determine the quantity (concentration) of protein in different solutions. The Coomassie Brilliant Blue dye was used to detect protein in solution. The red-orange color of the free floating dye in solution becomes blue when there is a detection of protein. The deeper blue the solution becomes, the more protein present. These solutions were then run through a spectrophotometer in order to read the absorbance of light in these blue solutions. Figure 2 shows how as more BSA protein

is added, the absorbance values become higher and higher. This correlates to the fact that more protein added causes a deeper blue colored solution. By having an absorbance value, a concentration could be calculated. The information shown in Table 2 shows the different fractions collected from lab 9 where a sample was taken from each, diluted, and then an absorbance value was read. V.

Discussion The predicted elution order was supported by the data. Due to the difference in hydrophobicity of each protein, each eluted at different buffers when they were introduced. The buffers act as a sort of unfolding mechanism where the hydrophobic interior of the protein can then interact with the surrounding substances and then elutes....